The ability to isolate and manipulate nucleic acid sequences encoding polypeptides has greatly increased research efforts into improved means for expressing these proteins in both natural and foreign hosts. Due to their ready availability, easy manipulation, and economy of use, unicellular microorganisms (e.g., bacteria and yeast) have been studied and utilized extensively for the production of such polypeptides.
When employing unicellular organisms, it is desirable to enhance the production of the polypeptide product of interest, with minimal interference on the production of other materials necessary for the cell's growth and/or maintenance. This permits maximal production of the desired products over an extended time period, with concomitant cost benefits. For these and other reasons, methods have been devised for selectively enhancing polypeptide expression in bacterial hosts.
One technique has been to couple the protein of interest to a promoter that is recognized by the host and allows for controlled regulation of efficient transcription of the gene encoding the polypeptide. Frequently, a "high-producing" host promoter is used, i.e., one associated with the natural production of a polypeptide that formally comprises a high percentage of the total protein of the host (or at least that provides a high transcription turnover rate). Typically, inducible promoters are preferred, because they permit expression of the gene under control in the presence of an inducing agent.
Generally, inducible promoters are useful only to the extent that the regulatory circuit and its components are understood and further to the extent that the components do not cross-react with other promoter or suppressor systems. Thus, well-defined and highly specific regulatory circuits, particularly promoter and regulatory components, have significant utilities.
In parallel to the above, the expanding research base concerning plant and related bacterial physiology has resulted in the development of various new agents active in plant nutrition, growth and protection (e.g.,pesticides, growth regulators, including hormones, herbicides, etc.). Presently, for crops such new agents (as well as those previously developed) are usually applied by spraying or irrigating the materials on most, if not all, of the field where the crops are grown. Frequently, as the agent need only interact with a certain portion of the plant (e.g., the roots) to be effective, this bulk application results in substantial waste. Also, in some cases, bulk application can actually prove to be harmful, when, for example, pesticide levels surpass safe limits.
The ability of certain bacteria in the gram-negative group Rhizobium to form nodules on the roots of plants, has provided a new potential avenue for selectively introducing agents into plants. The bacteria invade the roots, multiply and eventually inhabit cells of the nodules as intracellular symbionts. This invasion capability for Rhizobium essentially extends through one family of plants, the Leguminosae, which includes such important crops as soybean, alfalfa, clover, beans, garden peas, peanuts, cowpeas, etc. However, to effectively utilize this capability for introducing additional desired agents requires increased understanding of the genetics of nodulation.
Thus, there exists a significant need for additional and improved means for applying agents to crops and other plants of .interest. Further, there exists a significant need for the development of defined and specific inducible promoter systems for use in bacteria such as those capable of forming root nodules. The present invention fulfills these needs.